Research Article Effect of Creatine Monohydrate ...downloads.hindawi.com/journals/tswj/2013/286075.pdfblood of creatine monohydrate treated and untreated albino mice ( Table )

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013, Article ID 286075, 6 pageshttp://dx.doi.org/10.1155/2013/286075

Research ArticleEffect of Creatine Monohydrate Supplementationon Various Hematological and Serum Biochemical Parametersof Male Albino Mice following Neonatal Hypoxia-IschemiaEncephalopathy

Shahid Iqbal,1 Nabia Nazir,1 Quratulane Gillani,1 Atif Akbar,2 and Furhan Iqbal1

1 Zoology Division, Department of Zoology, Institute of Pure and Applied Biology, Bahauddin Zakariya University,Multan 60800, Pakistan

2Department of Statistics, Bahauddin Zakariya University, Multan 60800, Pakistan

Correspondence should be addressed to Furhan Iqbal; [email protected]

Received 2 August 2013; Accepted 25 August 2013

Academic Editors: F. A. Simmen and A. Surguchov

Copyright © 2013 Shahid Iqbal et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. Present study was designed to report the effect of 2% creatine monohydrate supplementation for 8, 12 and 15 weekson hematology and serum biochemical profile of male albino mouse following hypoxic ischemic insult on postnatal day 10.Methods. 66 Blood samples (2% creatine monohydrate supplemented (𝑁 = 34) and unsupplemented (𝑁 = 32)) were analyzed forvarious hematological (blood glucose, packed cell volume, total WBC count, total RBC count) and serum biochemical parameters(cholesterol, AST, ALT, HDL, LDL, total protein, triglycerides). Results. ALT had higher concentrations in mice feeding on normaldiet for 8 (𝑃 > 0.01) and 12 weeks (𝑃 > 0.01) following asphyxia and in 12 weeks treatment without asphyxia (𝑃 = 0.006) whencompared with the creatine supplemented mice. LDL (𝑃 = 0.011) and cholesterol (𝑃 > 0.01) had higher concentrations in mice onnormal diet for 12 weeks following hypoxia ischemia. Cholesterol (𝑃 > 0.01) in 12 and glucose (𝑃 = 0.006) in 15 week treatmentgroup had significantly lower concentrations in creatine supplemented male albino mice when compared with untreated groupfollowing hxpoic-ischemic insult. Conclusion. We concluded that creatine supplementation following hypoxic ischemic insult helpsin maintain the normal blood chemistry.

1. Introduction

Hypoxic ischemic encephalopathy (HIE) is a common causeof neonatal brain injury with an incidence rate of 2 in 1000term infants in the developed world. 15–20% of infants withHIE die in neonatal period, and 25–30% of survivors areleft with permanent neurodevelopmental abnormalities [1].Hypoxic ischemic injury in neonates is caused by variety ofconditions such as birth asphyxia, cardiac arrest, or respira-tory failure. Lack of regenerative ability in the central nervoussystem after injury is considered to be its fundamental cause[2]. HIE can lead to postneonatal neurological sequelae, thatis, cerebral palsy, mental retardation, learning disability, andepilepsy [3–6]. Data from Pakistan has shown that it is one ofthe leading causes for admission in a neonatal unit [7]. It has

also been shown that most causes of neonatal morbidity inPakistan are preventable [8].

Creatine (Cr) and phosphocreatine (PCr) play an essen-tial role in the storage and transmission of phosphate boundenergy [9]. Creatine is an amino acid derivative that isfound naturally, to small extent, in human body [10]. Liver,pancreas, and kidney synthesize approximately 2% of thetotal body creatine while 98% of creatine is taken with food.Creatine is naturally present in meat and to some extentin green leaved vegetables. 60% of creatine found in thebody is in the form of creatine phosphate [11]. Creatine hasbeen shown to be neuroprotective in various neurologicalconditions [12]. Acute oral creatine supplementation reducesmental fatigue on a serial calculation task [13]. Creatine showsprotective effects inmodels of acute neuronal damage, such as

2 The Scientific World Journal

traumatic brain injury [14] and stroke [15]. In animal modelsof neurodegenerative disease, creatine monohydrate supple-mentation has been shown to improve brain functioning,reduce oxidative stress, and attenuate neuronal degeneration[16–18]. Despite of extensive studies on creatine and its neu-roprotective effects, little information is available regardingthe effect of creatinemonohydrate supplementation on bloodchemistry following hypoxic ischemic encephalopathy.

Present study was conducted to study the effect of 2%creatine monohydrate supplemented or normal rodent dieton selective parameters of albino mice blood profile undernormal conditions as well as following neonatal hypoxicischemic encephalopathy (HIE) at postnatal day 10.

2. Materials and Methods

2.1. Experimental Animals. Male albino mice were used asexperimental animals and maintained in cages filled withwood chips at animal facility, Institute of Pure and AppliedBiology (IPAB), Bahauddin Zakariya University, Multan,Pakistan. In breeding colony, standard mouse diet and waterwere available. Room temperature was maintained at 22 ±1∘C. The light/dark rhythm was maintained at 14 : 10 hours.The room was illuminated with artificial light at an intensityof about 200W from 8 a.m. to 7 p.m. All the experimentalprotocols and mouse handling procedures were approvedby the ethical committee of IPAB at Bahauddin ZakariyaUniversity Multan.

2.2. Murine Model of Hypoxic-Ischemic Encephalopathy. Onpostnatal day 10, corresponding to a brain development offull-term (40 weeks) gestational period in human fetus, malepups were anesthetized using isoflurane (3%) inhalation. Aright lateral neck incision was made, and the right commoncarotid artery was ligated using polypropylene falcon USP6 suture. Pups were kept on a hot plate with constant 36∘Ctemperature during the surgery.The entire surgical procedurewas completed within 10 minutes. Mice were then placed for25 minutes in a hypoxic chamber with constant flow of 8%oxygen balanced with nitrogen. The hypoxic chamber waskept inwater bath tomaintain the ambient temperature insidethe chamber at 36∘C. Following hypoxic exposure, pups werereturned back to their mothers for recovery.

2.3. Experimental Design. On the 18–20 days of life, exper-imental animals were separated from their parents anddivided into four experimental groups. In first experimentmice were feed with diet supplemented with 2% creatinemonohydrate, while control group were fed with standardrodent diet for 8 weeks following hypoxic ischemic insult onpostnatal day 10. In second and third experimental series, thesame design was followed asmentioned above expect that theduration of 2% creatine monohydrate supplementation andstandard diet supplementation was 12 and 15 weeks, respec-tively, after weaning. A separate fourth cohort of animals wasused to study the effect of 2% creatine monohydrate supple-mentation for 12 weeks on hematology and serum biochem-istry of male albino mouse without hypoxic ischemic insult.

2.4. Blood and Serum Collection. At the end of each experi-ment, mice were anesthetized with 3% isoflurane, and bloodwas sampled either from retro-orbital sinus or through directcardiac puncture. Blood was divided into two parts; one forthe study of hematological parameters and second for serumbiochemical profiling.

2.5. Hematological and Serum Biochemical Analysis. Hema-tological parameters (blood glucose level, mean corpuscularvolume, packed cell volume, total red, and white bloodcell count) and serum biochemical parameters (cholesterol,aspartate transaminase (AST), alanine transaminase (ALT),high density lipoprotein (HDL), low density lipoprotein(LDL), total protein, and triglycerides) were determined intreated and untreated male albino mouse blood samples byusing Hitachi 902 automatic analyzer (Japan).

2.6. Statistical Analysis. All the data is expressed as mean ±standard deviation. Statistical package Minitab (version 13,Pennsylvania) was used for the analysis of results. Two-sample 𝑡-test was applied to compare various parametersof hematology and serum biochemical profile of albinomouse between 2% creatine monohydrate treated and theirrespective untreated controls following hypoxia ischemiaencephalopathy. Same statistical test was applied to comparevarious studied parameters between 2% creatine monohy-drate treated and untreated male albino mouse.

3. Results

3.1. Effect of 8 Week-Creatine Monohydrate Supplementationon Blood Profile of Albino Mouse after HIE. Results indi-cate that all other studied parameters remained unaffectedwhen compared with 2% creatine monohydrate treated anduntreated albino mice after 8 weeks of supplementationexcept ALAT concentration which was significantly higher(𝑃 > 0.01) in creatine untreated group (Table 1).

3.2. Effect of 12 Week-Creatine Monohydrate Supplementationon Blood Profile of Albino Mouse after HIE. When hemato-logical and serum biochemical parameters were comparedbetween mice supplemented with 2% creatine monohydratefor 12 weeks and their untreated control after HIE. It wasobserved that Cholesterol (𝑃 > 0.01), ALAT (𝑃 > 0.01) andLDL (𝑃 = 0.01) concentrations were significantly different inblood of creatine monohydrate treated and untreated albinomice (Table 2).

3.3. Effect of 15 Week-Creatine Monohydrate Supplementationon Blood Profile of Albino Mouse after HIE. Glucose (𝑃 =0.006) concentration was found significantly different inblood of 2% creatine monohydrate treated and untreatedalbino mice after 15 weeks of supplementation (Table 3).

3.4. Effect of 12 Week-Creatine Monohydrate Supplementationon Blood Profile of Albino Mouse without HIE. LDL (𝑃 =0.006) and ALAT (𝑃 = 0.006) concentrations were signifi-cantly different in blood of male albino mouse treated with

The Scientific World Journal 3

Table 1: Comparison of various hematological and serum biochemical parameters between 2% creatine monohydrate treated and untreated(control) male albino mice for 8 weeks following hypoxia and ischemia: data is expressed as mean ± standard deviation.

Parameters Creatine monohydrate treated group Untreated control group𝑃 value

Mean ± SD Range Mean ± SD RangeHematological profile

WBC (×109/L) 6.006 ± 2.648 11.5–2.65 5.833 ± 3.099 11.3–2.5 0.901 nsRBC (×1012/L) 4.96 ± 2.62 1.39–10.11 5.28 ± 1.14 4.0–7.18 0.748 nsPCV% 25.8 ± 11.1 11.3–38 27.53 ± 8.27 12.2–42.5 0.731 nsGlucose (mg/dL) 161.1 ± 38.4 98–214 136.2 ± 19.1 101–156 0.142 nsMCV (fp) 84.6 ± 85.4 29.6–258.2 53.0 ± 15.4 27.8–78.2 0.370 ns

Serum biochemical profileTotal protein (g/dL) 38.00 ± 9.17 28–46 53.00 ± 1.00 53-54 0.106 nsTriglycerides (mg/dL) 234 ± 125 130–393 280 ± 140 201–410 0.646 nsCholesterol (mg/dL) 824 ± 196 489–984 579 ± 208 411–811 0.174 nsHDL (mg/dL) 49.00 ± 3.00 46–52 49 ± 1.00 48–50 1.000 nsLDL (mg/dL) 662 ± 237 411–883 482 ± 222 283–722 0.408 nsALAT (𝜇/L) 23.67 ± 9.07 14–32 248.67 ± 8.08 240–256 𝑃 > 0.001

∗∗∗

ASAT (u/L) 156.0 ± 16.4 138–170 142.0 ± 20.0 122–162 0.417 ns𝑃 < 0.05 = non-significant (ns), ∗∗∗𝑃 > 0.001 = highly significant.


Parameters Creatine monohydrate treated group Untreated control group𝑃 value


WBC (×109/L) 5.91 ± 2.85 3.4–10.7 9.170 ± 6.518 5.15–20.5 0.347 nsRBC (×1012/L) 6.79 ± 2.74 4.59–10.86 6.76 ± 2.60 3.95–9.61 0.985 nsPCV% 27.83 ± 7.41 19.2–39.5 25.03 ± 5.42 20–33 0.492 nsGlucose (mg/dL) 169.5 ± 78.8 101–280 115.8 ± 40.2 60–162 0.188 nsMCV (fp) 47.1 ± 22.8 19.8–80.4 40.3 ± 11.9 26–50.6 0.544 ns

Serum biochemical profileTotal protein (g/dL) 3.2 ± 0.1 3.1–3.3 34.7 ± 27.3 17.4–66.1 0.184 nsTriglycerides (mg/dL) 427 ± 314 65–616 305.3 ± 68.0 228–356 0.579 nsCholesterol (mg/dL) 358.4 ± 32.4 318–394 753.3 ± 33.1 722–788 𝑃 > 0.001

∗∗∗

HDL (mg/dL) 20.67 ± 4.04 17–25 35.0 ± 20.0 18–57 0.347 nsLDL (mg/dL) 240.3 ± 72.5 173–317 656.7 ± 21.9 632–674 0.011∗

ALAT (𝜇/L) 72.33 ± 6.81 67–80 191.7 ± 10.4 180–200 𝑃 > 0.001∗∗∗

ASAT (u/L) 231.7 ± 10.4 22–240 161.7 ± 22.5 140–185 0.639 ns𝑃 < 0.05 = non-significant (ns), ∗𝑃 > 0.05 = Least significant, ∗∗∗𝑃 > 0.001 = highly significant.

2% creatine monohydrate for 12 weeks when compared withcreatine untreated male albino mice (Table 4).

4. Discussion

The decreased tissue oxygen supply below the normal levelis called hypoxia while reduced blood flow to organ whencerebral artery is blocked by a clot is called ischemia.During hypoxia, reactive oxygen specie (ROS) formation isincreased and leads to stress oxidative condition [19]. Bloodis an important medium in assessing the health [20], and

physiological and pathological conditions of animals can beassessed by the evaluation of hematological and biochemicalanalyses of the blood [21]. The present study was designedto evaluate the effect of 2% creatine monohydrate supple-mentation on the blood chemistry and hematology of albinomouse under hypoxic ischemic and nonhypoxic ischemicconditions.

Our results indicated that creatine supplementation afterhypoxic and ischemic insult has no effect on studied hemato-logical parameters of blood, as there was no significant differ-ence in hematological parameters when compared with 2%



Parameters Creatine monohydrate treated Group Untreated control group𝑃 value


WBC (×109/L) 9.94 ± 5.64 3.2–17.4 9.94 ± 1.66 7.4–11.8 0.806 nsRBC (×1012/L) 7.512 ± 0.963 6.25–9.03 7.55 ± 2.98 2.77–10.58 0.977 nsPCV% 31.5 ± 13.1 13.5–52.3 29.11 ± 9.66 14.2–41.6 0.681 nsGlucose (mg/dL) 177.8 ± 18.4 150–205 211 ± 21 176–245 0.006∗∗

MCV (fp) 42.5 ± 17.2 15–71.6 53.9 ± 47.9 13.4–150 0.570 nsSerum biochemical profile

Total protein (g/dL) 3.83 ± 0.93 2.5–5 2.80 ± 1.66 1.6–4.7 0.423 nsTriglycerides (mg/dL) 210 ± 146 59–399 331 ± 79.2 240–399 0.188 nsCholesterol (mg/dL) 178 ± 131 75–399 315 ± 192 129–513 0.356 nsHDL (mg/dL) 31.00 ± 3.00 28–34 28.00 ± 8.00 20–36 0.605 nsLDL (mg/dL) 69.7 ± 46.0 25–117 221 ± 169 60–397 0.110 nsASAT (𝜇/L) 227.7 ± 60.1 162–280 608 ± 330 144–889 0.110 nsALAT (𝜇/L) 40.8 ± 30.6 20–86 100.5 ± 76.2 42–208 0.242 ns𝑃 < 0.05 = non-significant (ns), ∗∗𝑃 > 0.01 = significant.

Table 4: Comparison of various hematological and serum biochemical parameters between 2% creatine monohydrate treated and untreated(control) male albino mice for 12 weeks: data is expressed as mean ± standard deviation.

Parameters Creatine monohydrate treated group (𝑁 = 10) Untreated control group (𝑁 = 10)𝑃 value


WBC (×109/L) 4.3 ± 1.5 2.5–8.5 8.3 ± 2.6 4.6–13.3 0.302 nsRBC (×1012/L) 5.69 ± 3.14 2.9–13.8 5.85 ± 2.67 2.1–10.5 0.903 nsPCV% 25.13 ± 5.58 17.3–34.8 28.0 ± 10.1 11.4–45.5 0.467 nsGlucose (mg/dL) 146.4 ± 23.2 107–193 156.1 ± 60.5 78–289 0.659 nsMCV (fp) 55.1 ± 27.3 12.3–97.2 61.0 ± 45.3 20.1–150 0.740 ns

Serum biochemical profileTotal protein (g/dL) 5.27 ± 1.23 3.9–6.3 40.9 ± 27.9 12.5–78.8 0.084 nsTriglycerides (mg/dL) 226.3 ± 16.2 216–245 395.0 ± 93.8 334–503 0.092 nsCholesterol (mg/dL) 376 ± 283 176–791 765 ± 118 629–839 0.068 nsHDL (mg/dL) 35.75 ± 9.22 23–45 20.67 ± 2.08 19–23 0.051 nsLDL (mg/dL) 152.6 ± 64.7 735–742 666 ± 107 94–222 0.006∗∗

ALAT (𝜇/L) 55.33 ± 5.03 50–60 81.00 ± 3.61 78–85 0.006∗∗

ASAT (u/L) 215.7 ± 46.7 165–257 157.33 ± 6.35 150–161 0.165 ns𝑃 < 0.05 = non-significant (ns), ∗∗𝑃 > 0.01 = significant.

creatine monohydrate supplemented and untreated controlgroup after 8, 12, and 15 weeks of 2% creatine monohydratesupplementation (Tables 1, 2, and 3). Similar results wereobtained in nonhypoxic ischemic group when hematologicalparameters were compared in male albino mouse on normalrodent diet with mouse supplemented with 2% creatinemonohydrate for 12 weeks (Table 4). Our results are in agree-ment with Eijnde et al. [22] who had reported that all valuesof blood hematology in human males following creatine

supplementation remained within the normal clinical rangethroughout the study with no significant difference.

In 2% creatinemonohydrate supplemented albinomouse,for 15 weeks, after hypoxic ischemic insult, blood glucoselevel was lower (177.8 ± 18.4) as compared with the creatineuntreated control group (211 ± 21), and the difference wasstatistically significant (𝑃 = 0.006) (Table 3). Decreasedglucose level after creatine supplementation was possibly dueto the fact that creatine supplementation leads to increased

The Scientific World Journal 5

insulin production confirming the findings of Rooney etal. [23] who had reported that chronic supplementation ofcreatine leads to hyper secretion of insulin decreasing theblood glucose concentrations.

Our results revealed that mouse exposed to hypoxic-ischemic insult had high serum cholesterol level whencompared with non-hypoxic ischemic group. Our resultsare consistent with the results of Li et al. [24, 25] whohad reported that hypoxia is one of the key mechanismwhich leads to the hyperlipidemia. Similar results were alsodocumented by Savransky et al. [26] as they observed thathypoxia raises serum cholesterol and LDL-C levels in micefed on a regular diet. In creatine supplemented hypoxic-ischemic mouse blood cholesterol level was lower (358.4 ±32.4) as compared with normal diet fed controls (753.3 ±33.1), and this difference was statistically highly significant(𝑃 = 𝑃 > 0.01) (Table 2). Earnest et al. [27] reported effect ofCr supplementation on the plasma lipid profile in humanmales and females and observed that individuals with highbasal cholesterol levels exhibited a reduction in blood totalcholesterol following creatine supplementation.

Our results indicated that mouse that suffers fromhypoxia and ischemia had high ALAT level as comparedwith their uninsulted control group. Increased ALAT levelafter hypoxia was also reported by Savransky et al. [26] whileinvestigating the effects of hypoxia on the liver in the absenceof obesity. Statistically significant difference in ALAT levelwas observed in hypoxic ischemic mouse after 8 weeks (𝑃 =𝑃 > 0.01) (Table 1) and 12 weeks (𝑃 = 𝑃 > 0.01) (Table 2) ofcreatine supplementation as compared with their normal dietfed mouse. ALAT variation was also observed in the groupwithout hypoxia and ischemia, In creatine supplementedmouse ALAT level was lower (55.33 ± 5.03) as compared withuntreated control group (81.00 ± 3.61), and this result wasstatistically significant (𝑃 = 0.006) (Table 4) indicating thatcreatine supplementation improves the liver functioning inalbino mouse.

5. Conclusion

Our results indicated that creatine supplementation afterhypoxic and ischemic insult has no effect on studied hema-tological parameters of blood while serum biochemicalprofile is significantly affected by hypoxia ischemia, and increatine monohydrate treated groups, studied parametershad concentrations lower than untreated groups indicatingthat creatine supplementation maintains serum profile ashigher values of certain enzymes like ALAT are indicators ofabnormal physiology.

Conflict of Interests

The authors have no conflict of interests.

Authors’ Contribution

Shahid Iqbal and Nabia Nazir contributed equally to thispaper.

Acknowledgment

The authors are grateful to the Higher Education Commis-sion (HEC) of Pakistan for providing research grant for thisstudy under the Indigenous Ph.D. Scholarship Scheme.

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Research Article Effect of Creatine Monohydrate ...downloads.hindawi.com/journals/tswj/2013/286075.pdfblood of creatine monohydrate treated and untreated albino mice ( Table )